Purpose <p>This study aimed to evaluate the in vitro biological activities of the polysaccharide derived from <i>Opuntia stricta</i> (Haw.) Haw. pulp (POS), as well as the wound healing efficacy in diabetic rats.</p> Methods <p>The study involved a comprehensive evaluation of the antioxidant properties, ᾳ-amylase and acetylcholinesterase inhibitory potential, as well as the anti-inflammatory and anti-hemolytic effects of the POS. Additionally, the present work aimed to assess the wound healing efficacy of POS in a diabetic rat model.</p> Results <p>POS exhibited antioxidant activities, effectively neutralizing total antioxidants, nitric oxide radicals, and protecting DNA from damage. Furthermore, POS showed potent ᾳ-amylase inhibition with an IC₅₀ value of 1968 µg/mL and an acetylcholinesterase inhibition with an IC₅₀ value of 157.33 µg/mL. Moreover, the results demonstrated that POS showed potential anti-inflammatory activity, preserved the integrity of the erythrocyte membrane and inhibited hemolysis. The administration of POS to a diabetic wound model showed a substantial improvement in wound healing and expedited the closure of the wound site after 15 days of induction. Its wound healing effects are attributed to its ability to regulate the balance of inflammatory markers, inhibit oxidative stress parameters, and improve antioxidant enzyme activities. Histopathological examination also revealed reepithelialization and complete epidermal development in the treated wounds.</p> Conclusion <p>Overall, these results highlight POS as a promising and effective agent for skin wound healing, providing valuable insights for its potential therapeutic application.</p> Graphical Abstract <p></p>